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Abstract

Clonality analysis is used to test malignant transformation and tumour progression. X-chromosome linked clonality assays have been employed for this purpose, but are subject to certain technical limitations. This paper reviews the issues involved and the controls that are necessary to ensure valid interpretation of such analyses.
Editorial
Designing a molecular analysis of clonality in tumours
Salvador J. Diaz-Cano
Department of Histopathology and Morbid Anatomy, St Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London
E1 1BB, UK
Abstract
Clonality analysis is used to test malignant transformation and tumour progression. X-chromosome
linked clonality assays have been employed for this purpose, but are subject to certain technical
limitations. This paper reviews the issues involved and the controls that are necessary to ensure
valid interpretation of such analyses.
Keywords: clonality; X-chromosome; lyonization; neoplasia
Clonality is an essential attribute of neoplasms and its
analysis has been used to test malignant transforma-
tion and tumour progression [1,2]. Concordant pat-
terns of genetic markers (X-linked or not) in different
tumours suggest that a common progenitor contribu-
ted to those lesions and favour, therefore, a multifocal
rather than a multicentric origin. These shared genetic
alterations also suggest a common cellular origin for
biphasic neoplasms [3]. Saxena et al. recently reported
a monoclonal pattern in smooth muscle cells and blood
vessels of sporadic angiomyolipoma, while the adipose
tissue revealed a polyclonal pattern [4]. Based on these
®ndings the authors concluded that the polyclonal
adipose tissue is probably metaplastic or reactive.
This represents a good example of the application of
clonality in tumour biology.
However, some biological and technical issues arise
from this article. X-linked clonality assays are based on
DNA polymorphism and random X-chromosome
inactivation (XCI) in females. Those features enable
us to distinguish the maternally from the paternally
inherited X-chromosomes [1,5,6]. The mechanisms
leading to XCI have not been fully characterized, but
DNA methylation might maintain the inactive state,
once it is established during early embryogenesis.
These methylation patterns are then transmitted by
clonal inheritance through the strong preference of
mammalian DNA (cytosine-5)-methyltransferase for
hemimethylated DNA, involving the promoter regions
of alleles on the inactive X-chromosome only [7]. Since
XCI analysis is based on differential DNA methylation
of one allele from X-chromosome genes (e.g. human
androgen receptor gene), suboptimal enzymatic diges-
tion and abnormal methylation can result in changes
of clonality patterns.
According to Lyon's hypothesis, all but one X-
chromosomes in a cell are randomly inactivated during
early embryogenesis, when the primordial cell pool
may comprise as few as 16±30 cells [8]. Given that
small number of embryo-destined cells, it reasonable to
expect unequal numbers of paternally- and maternally-
inherited inactive X-chromosomes, although the X-
chromosome is randomly inactivated in each cell. The
average Lyonization ratio is close to 50 : 50 in large cell
populations, although individual variation has been
found [8]. Skewing towards one allele to an extent that
meets the criteria for clonal derivation is consistent
with early XCI during embryogenesis (Figure 1).
This ®nding leads us to consider the selection of
appropriate controls to assess the Lyonization ratio in
each female. This ratio can also vary from tissue to
tissue in the same individual, due to unequal splitting
of the cells derived from the primordial cell pool, or to
different methylation patterns in different tissues [6,9].
Controls for unequal Lyonization should thus ideally
be the most closely related tissue thought not to be
involved in the disease process. An essential require-
Figure 1. Methylation pattern of androgen receptor alleles in
control samples. Only polymorphic and polyclonal controls (two
allele bands in both undigested and digested samples) are
considered informative for clonality assays (lanes 1 and 2). The
remaining possibilities (lanes 3±8) should be excluded from
clonality analyses, due to either monoclonal origin of controls
(lanes 3±6) or absence of locus polymorphism (lanes 7and 8).
U=undigested sample: D=digested sample
Journal of Pathology
J Pathol 2000; 191: 343±344.
Copyright #2000 John Wiley & Sons, Ltd.
ment for clonality analysis is the identi®cation of a
polymorphic locus in the normal control (Figure 1). In
every case, the tumour sample must be compared with
matched controls from the same patient to test the
heterozygosity for the marker. Additionally, the indi-
vidual variability and tissue-related Lyonization ratio
require samples of close embryological origin. This
feature must be maintained in the digested sample in
those tests based on XCI (Figure 1).
Positive allelic imbalances are determined case-by-
case, using the skewed data normalized by the allele
ratio in matched controls [1,6]. Allelic imbalance
analysis is based on the allele ratio and requires
densitometric analysis of both allele bands. Therefore,
the allele ratio in the target DNA must be maintained
in the ampli®cation product, which has to avoid the
PCR plateau phase. At this level, any PCR ampli®ca-
tion bias should be considered, especially DNA
degradation of the larger allele in formalin-®xed,
paraf®n-embedded tissues and defective ampli®cation
of repetitive CG-rich sequences [10±12].
Early XCI occurs randomly and results in a chess-
board pattern of cells descended from a common
progenitor, which may grow together like a clone
(patch size mosaicism). This pattern represents an
example of tissue heterogeneity that can also be present
in tumours. Sample size is a limiting factor; the lower
the cell number, the higher the probability of mono-
clonal patterns based on patch size mosaicism. This
concept becomes particularly important in mixed
tumours, where multiple microdissected samples from
different tumour areas (i100 cells) and from controls
are required to address the question.
Monoclonal patterns support a neoplastic rather
than a reactive or hyperplastic process, but are not
diagnostic of it. Host cell contamination of tumour
samples could give false heterozygous results that
would require careful microdissection and microscopic
control of the sample collection. However, the pitfalls
mentioned above should be always excluded.
Some of these considerations do not appear to have
been addressed in the paper of Saxena et al. [4],
especially those concerning tests for digestion comple-
tion with restriction endonuclease; controls regarding
both tumour heterogeneity and their methylation
patterns; PCR bias in the ampli®cation of both alleles;
tumour heterogeneity and patch size mosaicism; and
the meaning of monoclonal and polyclonal patterns.
References
1. Diaz-Cano SJ, Blanes A, Wolfe HJ. PCR-based techniques for
clonality analysis of neoplastic progression. Bases for its
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344 Editorial
Copyright #2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 343±344.
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... The importance of such approach is highlighted by the observation of clustered populations within a tumor that differ in gene expression [133], as well as genetic composition [134]. However, unless large numbers of samples are provided for each tumor, this approach can easily fail to identify patches of genetically distinct cells [130][131][132]. On the other hand, larger samples, or pools of samples, lead to intermixing of small anatomically distinct units, which provides additional challenges in relation to distinguish distinct functional heterogeneity. ...
... The histopathological diagnosis should integrate molecular analysis (genome sequencing, transcriptome profiling) and protein expression profiling (especially analyses including next-generation sequencing (NGS) techniques) and be able to include gene signatures that are characteristic of a different prognosis or clinical treatment [55,70,92,94,130,[135][136][137] (Fig. 3). The incorporation of NGS and the development of new resources for the analysis of these big data, combining molecular and expression signatures, are becoming crucial for diagnosis [13,138]. ...
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... Only informative cases (balanced allele ratios in undigested and digested controls) were included in the final analysis (10,11,13,25,26,28). Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Co., St Petersburg, FL), considering only allele ratios greater than 4:1 in the normalized digested lanes evidence of monoclonality. ...
... In addition, technical aspects such as PCR bias against the larger allele could contribute to preferential amplification of the smaller allele. Our DNA extraction protocol included a long protein digestion and retrieved DNA of ϳ1 kb (data not shown), excluding degraded DNA as cause (13,22,25,28,44). Our PCR design also included long denaturation and extension in the first three cycles and 7-deaza-dGTP in the amplification mixture to avoid defective amplification of CG-rich sequences (Table 1) (10,13,24,25,40). ...
... The polyclonal pattern in patients with germline RET mutation (CCH-4) needs some considerations. XCI assays can result in polyclonal patterns if the restriction endonuclease digestion is incomplete or the target DNA is hypermethylated (10,11,13,25,28). Suboptimal enzymatic digestion may change the clonality pattern of monoclonal tissues (10, 28), but that possibility was excluded keeping internal controls of endonuclease digestion. ...
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... Neoplasms result from the progressive and convergent selection of cell populations for which clonality is still the hallmark indicative of acquired somatic mutations [7][8][9][10]. However, the molecular events during neoplastic transformation are not completely understood and remain essentially unknown, which leaves X-chromosome inactivation assays as the best molecular option, because this test is not based on any tumor-related genetic alteration [11,12]. ...
... Sample normalization was done in relation to the corresponding undigested sample and tissue controls. Only informative cases (2 different alleles in HhaI-undigested and HhaIdigested samples) were included in the final analysis [8,9,14,15,28]. ...
... Benign monoclonal adrenocortical lesions reveal simultaneous apoptosis downregulation and proliferation upregulation, and promote a stromal vascular reaction to support this demanding cell kinetics [14,28]. The inverted proliferation/ apoptosis relationship in monoclonal adrenocortical lesions also provides a functional basis for cellular selection, leading to clonal expansion (if proliferation predominates) or regression (if apoptosis dominates) [7,8,13,17,41,42]. The significantly increased rate of hypertetraploid cells (high 5cER correlated with monoclonal patterns) would support the presence of cycling tetraploid G 0 /G 1 cells, which suggests associated abnormalities in the anaphase checkpoint (Fig. 3). ...
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... Interpretation and inclusion criteria in each sample were achieved as follows: 7,11,21,22,26,27 (i) allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Corp., St Petersburg, FL, USA). For evidence of loss of heterozygosity (LOH) only allele ratios ‡ 4 : 1 in any TSG were considered; otherwise retention of heterozygosity (ROH) was assigned. ...
... For evidence of loss of heterozygosity (LOH) only allele ratios ‡ 4 : 1 in any TSG were considered; otherwise retention of heterozygosity (ROH) was assigned. 7,11 This ratio represents 80% of clonal cells in the sample and was used to increase the detection specificity; 22,26,28 (ii) additional allele bands present in tumour samples but not in the corresponding controls were considered evidence of somatic single nucleotide polymorphism (SNP) by PCR ⁄ denaturing gradient gel electrophoresis. 7,19,22,29 dna sequencing ...
... Technical reasons were excluded. The sensitivity threshold of our optimized protocol was 1% for positive detection, 7,11,22,26 which applied to 100+ cell samples would result in false-negative results for DNA samples smaller than one cell equivalent. This is probably clinically irrelevant and frequently related to contamination. ...
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... MSI can be defi ned as a change in any DNA sequence length due to either insertion or deletion of repeating units in a microsatellite within a tumor when compared to normal tissue [17,18] . The tests must be run with appropriate controls (known positive and negative controls along with the patient's normal tissue) [8,19] , which are extremely important due to the nonexceptional presence of extra-bands. The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient's normal tissue. ...
... (3) PCR bias against one allele (especially the larger one in a pair) can result in preferential amplifi cation of the other allele (usually the smaller in a pair), which is the so-called artifactual allele dropout [22,23] . An appropriate extraction method, providing DNA of quality [24] , and PCR designs including both long denaturation and extension in the fi rst three cycles and 7-deaza-dGTP in the amplifi cation mixture to improve the amplification of CG-rich DNA regions, will be reasonably helpful in avoiding that bias [8,19,21,23,25] . (4) The number of polymorphic DNA regions agreed to at the NCI consensus conference includes a primary panel of at least 2 mononucleotide and 3 dinucleotide microsatellites, along with 19 alternate loci (both mono-and dinucleotides) [26] . ...
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... The average Lyonization ratio is close to 50:50 in large cell populations, although individual variation has been found (23), resulting in skewing toward one allele. This is the reason for using as controls for unequal Lyonization the most closely related tissue thought not to be involved in the disease process (14). ...
... No single genetic alteration of TSG proves by itself that a given proliferation is monoclonal: The LOH for that particular marker informs only on clonal expansion and cellular selection in genetically heterogeneous tumor cell populations. Only the accumulation of genetic lesions in TSG supports a monoclonal origin of tumors (15), especially if multiple samples from the same tumor show concordant genetic alteration (14,18) (see Methodological Aspects). ...
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... Interpretation and inclusion criteria in each sample were previously reported (11,13,(21)(22)(23). Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus Corp., St. Petersburg, FL), considering evidence of only LOH allele ratios of 4:1 or more in any TSG; otherwise, retention of heterozygosity (ROH) was assigned (13,22). ...
... Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus Corp., St. Petersburg, FL), considering evidence of only LOH allele ratios of 4:1 or more in any TSG; otherwise, retention of heterozygosity (ROH) was assigned (13,22). This ratio represents 80% of clonal cells in the sample and was used to increase the detection specificity (10,11,23). Additional allele bands present in tumor samples, but not in the corresponding controls, were considered evidence of somatic SNP by PCR/denaturing gradient gel electrophoresis (DGGE) (13). ...
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... The opposite profile was observed in carcinomas, confirming the kinetic equivalency between superficial ⁄ internal and deep ⁄ peripheral compartments of neoplasms from luminal and solid organs. [5][6][7] Cell kinetics (proliferation and apoptosis) represents the basic mechanism leading to clonal expansion and tumour growth [29][30][31][32][33][34] and contributes to the theory of tumour cell topographical segregation. [5][6][7]13,14 High proliferation rates in ICs of FTA and FTHN support this theory. ...
... High-grade neoplasms show high apoptotic indices, which should be the result of the accumulation of genetic mutations reaching lethal levels for the cell. [31][32][33] Follicular tumour progression correlates with up-regulation of proliferation and relative down-regulation of apoptosis in PCs, suggesting survival and replication of genetically damaged cells leading to the accumulation of genetic mutations: 5-7,13,14 high cell proliferation would transfer mutations to daughter cells, while reduced apoptosis would allow cells carrying mutations to complete the cell cycle. ...
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... Only informative cases (2 different alleles in control samples) were included in the final analysis and were interpreted as described. 2,12,13,30 Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus, St Petersburg, FL). Only allele ratios of 4:1 or more in any TSG were considered evidence of loss of heterozygosity (LOH); otherwise retention of heterozygosity was assigned. ...
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... The heterogeneous distribution and clustering of apoptotic cells (significantly higher ISEL-index SD) in the superficial compartments also support a sort of clonal origin for these apoptotic cells, most likely due to the accumulation of genetic abnormalities reaching cytologically lethal levels. 32,41 Likewise, the coexistence of genetic alterations in MCC supports a key role in tumorigenesis, the topographic heterogeneity resulting from the accumulation of genetic damage, partially explained by TP53 overexpression in these neoplasms. 6,37,42 This TP53 overexpression frequently correlates with mutated TP53 that partially blocks apoptosis and allows accumulation of mutations. ...
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